Stabilized Ultrasound Contrast Agent

ABSTRACT

Provided is a particle-stabilized ultrasound contrast agentn (UCA) and methods of preparing same. Also provided is a method for preparing a lyoprotected UCA, a freeze-dried lyoprotected UCA, and a reconstituted freeze-dried lyoprotected UCA.

BACKGROUND OF THE INVENTION

Ultrasound contrast agents are typically gas-filled microbubbles thatare administered intravenously to the systemic circulation. Microbubbleshave a high degree of echogenicity, that is, they reflect the ultrasoundwaves. The echogenicity difference between the gas in the microbubblesand the soft tissue surroundings of the body is immense. Thus,ultrasonic imaging using microbubble contrast agents enhances theultrasound backscatter, or reflection of the ultrasound waves, toproduce a unique sonogram with increased contrast due to the highechogenicity difference. Contrast-enhanced ultrasound can be used toimage blood perfusion in organs, measure blood flow rate in the heartand other organs, and has other applications as well.

A series of surfactant-based UCA, composed of sonicated mixtures ofnon-ionic surfactants which self-assemble around a gaseous core, havebeen developed (U.S. Pat. No. 5,352,436). One particular agent, ST68,consists of Span 60 and Tween 80 12 filled with octafluoropropane (a PFCgas) (Basude et al., 2001, Ultrasonics, 39, 437-44). This agent canconsistently be produced with a mean size of 1.5 to 2 μm and producesover 20 dB enhancement for doses less than 100 μl/l in vitro (Basude etal., 2000, Ultrasound Med. Biol., 26, 621-8) and 0.05 ml/kg in vivo(Forsberg et al., 1996, 1996, IEEE Ultrasonics Symp., 2, 1337-40).However, further development of this agent is hampered by the fact thatit consists of an aqueous suspension of bubbles, which have limitedstability with time (less than a week at 4°18 C). An ideal UCA has beendescribed as being stable at room temperature for at least 6 months(Wang et al., 1996, J. Phys. Chem., 100, 13815-21).

The technique of freeze-drying, or lyophilization, has been implementedto increase the shelf-life and stability of vaccines, viruses, andproteins in pharmaceutical production (Jennings, 1999, Lyophilization:Introduction and Basic Principles. Interpharm. Press. Denver, Colo.) andin liposomes as drug carriers with and without acoustic reflectivity(i.e. increased echogenicity) (Huang et al., 2002, Cell Molec. Biol.Letters, 7, 233-5; Hua et al., 2003, Drying Technol., 21, 1491-505).However, this process generates stresses during the freezing and dryingstages which could destabilize the suspension and destroy the bubbles(Abdelwahed et al., 2006a, Euro. Pharm. Biopharm., 63, 87-94). Someagents, such as liposomes, require the addition of cryoprotectants toaid stability during freezing (Ozer et al., 1988, Acta Pharm. Technol.,34, 129-39) or lyoprotectants to help prevent structural and functionalintegrity loss that occurs during the drying process (Jennings 1999,supra). This is achieved by preventing fusion and aggregation duringfreeze-drying thus allowing for better reconstitution (Hua et al., 2003,supra). It has been suggested that the major damaging factors associatedwith freeze-drying liposomes are lipid-phase transition and fusion(Crowe et al., 1997, Cryobiol., 35, 20-30).

In order to be used safely in a clinical setting as well as to accessvarious biological compartments, a contrast agent must have a diameterless than 8 μm. Typically, it has been difficult to fabricate ultrasoundcontrast agents in the nanometer range that are as functionallyeffective as their micrometer counterparts. In addition, all microbubbleUCA suffer from a lack of stability, and being susceptible todegradation when freeze-thawed. Accordingly, there is an ongoing need inthe art for the development of stable ultrasound contrast agents in thesub-micron size-range. There is also a need for an ultrasound contrastagent that is not susceptible to freeze-drying degradation. The presentinvention fills this need.

BRIEF SUMMARY OF THE INVENTION

Provided is a freeze-dried ultrasound contrast agent (UCA) comprising atleast a first surfactant, a second surfactant and a saccharide. In anembodiment, the saccharide is selected from the group consisting ofglucose and trehalose.

Also provided is a reconstituted UCA, comprising a freeze-dried UCA andan excipient, wherein the freeze-dried UCA comprises at least a firstsurfactant, a second surfactant and a saccharide. In an embodiment, thesaccharide is selected from the group consisting of glucose andtrehalose.

Also provided a particle-stabilized ultrasound contrast agent (UCA)comprising at least a first surfactant and a second surfactant, whereinthe first surfactant is d-α-tocopheryl polyethylene glycol 1000succinate (TPGS), wherein said UCA further comprises a particulatematerial that stabilizes the UCA, wherein the diameter of said UCA isbetween 1 nm and 1 μm.

The surfactants of the freeze-dried UCA, reconstituted UCA orparticle-stabilized UCA can be selected from the group consisting ofSPAN, alkylphenol ethoxylate-based surfactants, alcohol ethoxylate-basedsurfactants, silicone-based surfactants, alkyl poly(ethylene oxide),alkylphenol poly(ethylene oxide), copolymers of poly(ethylene oxide) andpoly(propylene oxide), alkyl polyglucosides, fatty alcohols, cocamideMEA, cocamide DEA, and polysorbates. In some embodiments, the firstsurfactant is TPGS.

In some embodiments of the freeze-dried UCA or reconstituted UCA, theUCA is a particle-stabilized UCA.

Also provided is a method of making a lyoprotected ultrasound contrastagent. The method comprises the steps of a) preparing a UCA comprisingat least a first surfactant and a second surfactant; and b) adding alyoprotectant to the UCA to prepare a lyoprotected UCA, wherein thelyoprotectant is a saccharide. Optionally, the method can furthercomprise the step of c) freeze-drying the lyoprotected UCA, therebypreparing a freeze-dried UCA. In an embodiment, the saccharide isselected from the group consisting of glucose and trehelose.

A method of making a particle-stabilized ultrasound contrast agent (UCA)is also provided. The method comprises the steps of: a) mixing at leasttwo surfactants in 50 ml of water where one of the surfactants is TPGSand heating the mixture until both surfactants are dissolved; b) coolingthe mixture to room temperature while stirring rapidly until thedispersible waxy solid comes out of solution as fine particles; c)purging the mixture using a sterile filtered gas in an ice bath; d)sonicating the mixture at between 100-140 W for 1-5 minutes withconstant purging; e) placing mixture in a separation funnel with 50 mlPBS to allow effective separation of the bubbles; f) discarding lower 25ml of the solution and transferring the next 50-75 ml of solutionremaining is placed in a second separation funnel; and g) washing thebubbles of desired size, collecting them, and optionally, freeze dryingthem for storage.

In the methods of the invention, the surfactants can be selected fromthe group consisting of SPAN, alkylphenol ethoxylate-based surfactants,alcohol ethoxylate-based surfactants, silicone-based surfactants, alkylpoly(ethylene oxide), alkylphenol poly(ethylene oxide), copolymers ofpoly(ethylene oxide) and poly(propylene oxide), alkyl polyglucosides,fatty alcohols, cocamide MEA, cocamide DEA, and polysorbates. In someembodiments, the first surfactant is TPGS.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIGS. 1A and 1B depict exemplary surfactants. FIG. 1A depicts thestructure of SPAN 60, FIG. 1B depicts the structure of TWEEN80.

FIG. 2 is a schematic diagram depicting a previously proposed model formicrobubbles stabilized by SPAN 60 and TWEEN 80.

FIG. 3 is a schematic depicting the position of a small sphericalparticle at the interface for a contact angle θ (measured through theaqueous phase) of less than 90° (left) or great than 90° (right). Forθ<90°, solid-stabilized aqueous foam or o/w emulsions may form. Forθ>90°, solid-stabilized aerosols or w/o emulsions may form.

FIG. 4 is a schematic diagram depicting possible mechanisms of liquidfilm stabilized by: (a) a monolayer of particles; (b) a bilayer ofclose-packed particles and (c) a network of particle aggregated insidethe film.

FIGS. 5A-5C are a series of images depicting possible approaches toattach colloidal particles at gas-liquid interfaces by tuning theirsurface-wetting properties. FIG. 5A depicts stabilization of gas bubbleswith colloidal particles (the particle size is exaggerated for clarity).FIG. 5B depicts the adsorption of partially-lyophobic particles at thegas-liquid interface. FIG. 5C depicts the approaches used to tune thewetting properties of originally hydrophilic particles to illustrate theuniversality of the foaming method developed.

FIGS. 6A-6C are a series of images depicting the hierarchical featuresof particle-stabilized foams containing short amphiphilic molecules.High-volume macroscopic foams (depicted in FIG. 6A) with bubble sizeswithin the range 10-50 mm (depicted in FIG. 6B) are formed through theadsorption of submicrometer-sized colloidal particles at the air-liquidinterface (depicted in FIG. 6C). Particles attach at the air-waterinterface as a result of the surface hydrophobicity imparted by theadsorbed amphiphilic molecules, as indicated schematically in FIG. 6D.The confocal images shown in FIG. 6B and FIG. 6C were obtained afterdilution of concentrated foams (inset in FIG. 6B) containingfluorescently-labeled silica particles and hexylamine as amphiphile.

FIG. 7 depicts the molecular structure of d-α-tocopheryl polyethyleneglycol 1000 succinate (TPGS).

FIG. 8 is a graph depicting the normalized foam volume of TWEEN 80(solid squares) and the mixture SPAN 60 and TWEEN 80 at the molar rationof 20:80 (solid triangles) and 80:20 (solid diamonds) over time. Theinitial concentration of surfactant is 85.1 mM.

FIG. 9 is a schematic diagram depicting a model for the bubblesstabilized by SPAN and TWEEN.

FIGS. 10A and 10B are a series of graphs depicting particle sizedistribution (FIG. 10A) of the mixture of 2 mM of SPAN 60 with 1 mM(curve a), 0.5 mM (curve b) and 0.1 mM (curve c) of TWEEN 80 in 50 ml ofPBS and (FIG. 10B) the time response curve from in vitro ultrasoundtesting of microbubbles produced from the mixtures.

FIG. 11 is a graph depicting the time response curve for SE61 (soliddiamonds), nSE61 (solid triangles) and standard ST68 (solid squares).

FIG. 12 depicts a graph of recorded temperature readings of lyoprotected(100 mM) and control ST68 samples during freeze-drying. Glucose additionmaintained the sample at a constant temperature of −12° C. longer (7hours) than other excipients: 1 to 2 hours for trehalose or sucroseaddition, and mannitol addition having a steadily rising temperatureprofile.

FIG. 13 depicts a dose response curve for lyophilized ST68 in a varietyof 100 mM candidate lyoprotectant solutions. All sugar controls (e.g.,glucose alone) are dissolved in PBS, lyophilized, and PFC gas introducedbut do not contain any agent. Glucose and trehalose were significantlygreater than the control, the sucrose and the mannitol by the samedegree (*p<0.001, error bars=SEAM, f=5 MHz, 684 kPa, PRF=100 Hz).

FIG. 14 depicts a bar graph of the half life of lyophilized ST68 in avariety of 100 mM candidate lyoprotectant solutions tested at room (23°C.) and body (37° C.) temperature. ANOVA testing revealed ST68 withglucose at room temperature to be the only lyoprotected agentstatistically greater than that of the ST68 control and all otherlyoprotectants (*p<0.05, **p<0.01). (f=5 MHz, 684 kPa, PRF=100 Hz).

FIGS. 15A and 15B are a series of graphs depicting normalized timeresponse of lyophilized ST68 in 100 mM lyoprotectant solutions tested at23° C. (FIG. 15A) and 37° C. (FIG. 15B) temperature. ANOVA testingshowed ST68 with glucose at 23° C. to be the only lyoprotected agentstatistically greater than that of the ST68 control and all otherlyoprotected samples after 15 minutes (*p<0.05, **p<0.01) (f=5 MHz, 684kPa, PRF=100 Hz),

FIGS. 16A-16E are a series of Olympus BX50 PLM images taken at 20× oflyophilized samples of ST68 with and without candidate lyoprotectantexcipients (100 mM). Mannitol (FIG. 16D) shows a crystalline structurewhile the others excipients form glassy matrices. Control ST68 samples(FIG. 16E) contained no evidence of either an amorphous glassy matrix orcrystallization, being diluted 1:1 with PBS instead of a sugar solution.Visually, glucose protected ST68 (FIG. 16A) seems to form the mostintact glassy matrix with trehalose (FIG. 16B) and sucrose (FIG. 16C)have glassy spindles.

FIG. 17 depicts a dose response curve of lyophilized ST68 in variousglucose concentrations. No statistical differences measured (p>0.05).(f=5 MHz, 684 kPa, PRF=100 Hz).

FIG. 18 depicts a bar graph of half life data of lyophilized ST68 invarious glucose concentrations. No differences are statisticallysignificant (p>0.05), (f=5 MHz, 684 kPa, PRF=100 Hz).

FIG. 19 depicts a bar graph of maximum echogenicity and half life dataof ST68G-100 tested over a period of 3 months at the start of eachmonth. No statistical differences were measured (p>0.05). (f=5 MHz, 684kPa, PRF=100 Hz).

FIGS. 20A and 20B are a series of Zeiss Supra 50 S.E.M. images ofST68G-100 (FIG. 20A) and ST68 without lyoprotectant (FIG. 20B) taken at6,000× with an Oxford Energy Dispersive Microanalysis (EDS) set to 3.5kV and an aperture of 4 mm. (Size bar=2 μm),

FIG. 21 depicts a dose response curves of ST68G-100 in-vivo performed ona New Zealand white rabbit with a Sonix RP scanner on pulse Doppler modeat 5 MHz and a PRF of 6.7 kHz.

FIGS. 22A and 22B are a series of images of before (FIG. 22A) and 5seconds after (FIG. 22B) 0.1 ml/kg injection of ST68G-100 into a 3.3 kgNew Zealand white rabbit with enhancement lasting for at least 40seconds (Sonix RP scanner in pulse inversion mode at 5 MHz, PRF of 1kHz, −8 power).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to the discovery that an ultrasoundcontrast agent (UCA) comprising particle stabilized micro- and/ornanobubbles is more stable compared to an UCA without particles. Theinvention is further related to the discovery that certain saccharidescan function as lyoprotectants for an ultrasound contrast agentsubjected to a freeze-dry process. Specifically, the saccharide enablesreconstitution of freeze-dried UCA, while substantially preserving theechogenicity observed for non-freeze-dried UCA. Other advantages,including structural stability, storage stability and in vivo doseresponse are also observed.

Definitions:

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used.

As used herein, the term “hydrophilic-lipophilic balance” (“HLB”) is arelative measure of the ratio of polar and non-polar groups present in asurfactant.

As used herein, “ultrasound contrast agent” (“UCA”) refers tosurfactant-stabilized gas bubbles.

As used herein, “lyoprotectant” refers to a compound that minimizes orprevents structural and/or functional integrity loss of UCA that canoccur during the drying stage of a freeze-drying process.

As used herein, “cryoprotectant” refers to a compound that providesstability during the freezing stage of a freeze-drying process.

It is understood that any and all whole or partial integers between anyranges set forth herein are included herein.

Description:

In one embodiment, the present invention is an ultrasound contrast agent(UCA) comprising stabilized gas bubbles, wherein the gas bubble isstabilized by at least two surfactants, and further wherein one of thosesurfactants is d-u-tocopheryl polyethylene glycol 1000 succinate (TPGS),also known as vitamin E (FIG. 3). The UCA of the invention are less than8 micrometer (μm). A preferred size range for the UCA of the inventionis between about 1 nanometer (nm) to about 1000 nm, such as betweenabout 1 nm and about 100 nm, between about 100 nm and about 300 nm,between about 300 nm and about 500 nm, between about 300 nm and about800 nm, and between about 500 nm and about 1000 nm.

Surfactants useful in the practice of the invention are anybiocompatible surfactants known in the art including anionic, cationic,zwitterionic, and nonionic surfactants. In a preferred embodiment, theUCA comprises at least one nonionic surfactant. Preferably, the at leasttwo surfactants are both nonionic surfactants. When the surfactant isnonionic, the hydrophilic-lipophilic balance (HLB) of the surfactant isbetween about 6 and about 16. Non-limiting examples of nonionicsurfactants useful in the practice of the invention include, but are notlimited to, TPGS, SPAN (e.g., SPAN 40 and SPAN 60), alkylphenolethoxylate-based surfactants, alcohol ethoxylate-based surfactants,silicone-based surfactants, alkyl poly(ethylene oxide), alkylphenolpoly(ethylene oxide), copolymers of poly(ethylene oxide) andpoly(propylene oxide), alkyl polyglucosides, fatty alcohols, cocamideMEA, cocamide DEA, and polysorbates (e.g., TWEEN20 and TWEEN80).

In some embodiments, the UCA at least two non-ionic surfactants selectedfrom the group consisting of SPAN, TWEEN and TPGS. The possiblecompositions in these embodiments include combinations of SPAN/TWEEN,SPAN/TPGS and SPAN/TWEEN/TPGS. The different surfactants in variouscombinations, in various concentrations, and encapsulating differentgases (e.g., air, sulfur hexafluoride (SF₆) and perfluorocarbon, (PFC))will dictate the packing density and strength of inter-molecular forcesaround the gas. In one embodiment, the UCA comprises SPAN 60. SPAN 60 isdepicted in FIG. 1A. In one embodiment, the UCA comprises TWEEN80.TWEEN80 is depicted in FIG. 1B, TPGS is depicted in FIG. 7.

The microbubbles and nanobubbles comprising surfactants may bestabilized by the presence of a particulate material. In one embodiment,the particulate material is one of the surfactants. In otherembodiments, an exogenous particulate material in incorporated into theUCA. The exogenous particulate material is incorporated any time priorto the sonication step. Examples of particles that are useful in thepractice of the invention include, but are not limited to, solidsurfactant; quantum dots; colloidal gold; carbon nanotubes; carbonnanotubes containing drug; polystyrene; bucky balls; SPIO(superparamagnetic iron oxide); iron oxide; coated nanoparticlescontaining a drug; imaging agents such as gas; radiopaque species; MRcontrast agents such as Gd compounds; a pure drug in nanoparticulateform (e.g., by grinding); nanocapsules; hollow drug-containing contrastmedium containing (CT, MRI, Spect) particles with attached targetingagents such as antibodies, portions of antibodies, peptide sequencesetc.; viruses; and inactivated viruses. The particle suitable for use inthe invention ranges in size from about 1 to about 500 nm, dependingupon the size of the microbubbles and nanobubbles. The contact anglebetween the material of the particles and the fluid should be less than90 degrees, (See FIG. 3, left side).

In the following embodiments, the specific volumes recited areexemplary. However, the methods of the invention are not limited tothese volumes. In one embodiment, the method of making aparticle-stabilized UCA of the instant invention comprises the followingsteps:

-   -   1. At least two surfactants are combined in 50 ml of water where        one of the surfactants is TPGS. In one embodiment, the other        surfactant comprises a dispersible waxy solid such as SPAN 60 or        SPAN 40. The mixture is heated until both surfactants are        dissolved. The mixture may then be autoclaved.    -   2. The mixture is cooled to room temperature while stirring        rapidly to allow the dispersible waxy solid to come out of        solution as fine particles.    -   3. In an ice bath, the mixture is purged using a sterile,        filtered gas such as PFC or SF6.    -   4. The mixture is then sonicated at between 70 and 140 W and        preferably between 100 and 140 W for 1-5 minutes with constant        purging.    -   5. Contents of the beaker are then placed in a separation funnel        with 50 ml PBS to allow separation of the bubbles.    -   6. After sufficient time to allow separation as determined by        the skilled artisan, the lowest 25 ml of the solution (e.g.,        about 25% of total solution) is discarded and the next 50-75 ml        of solution remaining is placed in a second separation funnel.        The mixture remaining in the first funnel contains micron sized        bubbles. The mixture in the second funnel contains nano-sized        bubbles.    -   7. The mixture in both funnels is washed and the bubbles of the        desired size collected. The bubbles are optionally freeze dried        according to methods described elsewhere herein.

In another embodiment, the method is practiced as follows:

-   -   1. 1.5 g of sodium chloride and 1.464 g of a dispersible waxy        solid, such as SPAN 60 or SPAN 40, and 1.288 g of TPGS are added        to 50 mL of PBS and the mixture is stirred.    -   2. The mixture is slowly heated with continuous stirring to        bring to boiling, and dissolve the solid and TPGS.    -   3. The mixture is autoclaved with the stir bar in place for        20-35 min.    -   4. The mixture is allowed to cool to room temperature for 30-45        minutes while rapidly stirring so that the SPAN comes out of        solution as fine particles.    -   5. Two 125 ml sterile separation funnels are set up in that cold        room to cool to 4° C.    -   6. The beaker is placed in an ice bath and purged with sterile        filtered gas, such as PFC or SF₆ gas, until bubbles cover the        solution before sonication. When purging with gas, the tip that        supplies the gas will be in the solution.    -   7. The mixture is sonicated at between 70 and 140 W and        preferably between 100 and 140 W for 1-5 minutes (Misonix Inc.        CL4 tapped horn probe with 0.5″ tip, Farmingdale, N.Y.) with        constant purging using a steady stream of gas of choice. When        sonicating, the tip supplying the gas is not in the solution;        only the sonication probe will be submerged,    -   8. The contents of the beaker are poured into one of the        separation funnels (designated funnel A), along with by 50 mL        cold PBS. The separation funnel is place in the fridge for about        one hour, or long enough to allow separation of the bubbles        according to size to occur.    -   9. After about 1 to about 2 hours, the lower 25 ml of solution        is discarded and the next 50-75 ml of the solution is        transferred to the second cold separation funnel (designated        funnel B).

At this stage separation funnel A contains the majority of micron sizedbubbles and funnel B contains the majority of nano sized bubbles,

Funnel A

-   -   1) The contents of funnel A are washed twice with 50 ml PBS,        allowing 35-50 minutes to elapse between each wash for        separation of the bubbles according to size. After each wash,        the middle layer containing predominantly micron sized bubbles        is collected, and the lower portion of the mixture is discarded.

Funnel B

-   -   1) The contents of funnel B are washed twice with 50 ml PBS,        allowing about one hour to elapse between each wash for        separation of the bubbles according to size. Twenty ml of        solution is discarded from the bottom of the separating funnel.        After the second wash, 10 ml of nanobubbles is collected.

Both preparations are then taken forward for either freeze drying orstorage at 4° C. For cold storage, samples are either taken up intovacutainer tubes which are completely filled, or placed in glass vials,which are tightly capped only after the head space has been purged withthe filling gas (e.g., PFC or SF₆). These vials are stored at 4° C.

It will be understood by the skilled artisan that standard steriletechniques may be used throughout the procedure in order to produce asterile composition comprising the particle stabilized UCA of theinstant invention.

The particle-stabilized UCA may be freeze-dried according to methoddescribed elsewhere herein for storage and later reconstituted using apharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable carrier” means a chemical composition withwhich the active ingredient may be combined and which, following thecombination, can be used to administer the active ingredient to asubject.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to a subject where the subject is ahuman, it will be understood by the skilled artisan that suchcompositions are generally suitable for administration to animals of allsorts.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

A pharmaceutical composition comprising a UCA of the invention, such asa particle stabilizes UCA, may be administered to a subjectparenterally. As used herein, “parenteral administration” of apharmaceutical composition includes any route of administrationcharacterized by physical breaching of a tissue of a subject andadministration of the pharmaceutical composition through the breach inthe tissue. Parenteral administration thus includes, but is not limitedto, administration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration is contemplated to include, but is not limited to,intravenous administration.

In another embodiment, the invention provides a method for freeze-dryingsurfactant-stabilized gas bubbles while substantially preserving theechogenicity and stability of the bubbles. Also provided arefreeze-dried surfactant-stabilized gas bubbles and reconstitutedfreeze-dried surfactant-stabilized gas bubbles.

The method comprises adding a saccharide to a UCA prior to freeze-dryingthe UCA. The saccharide is preferably one of glucose or trehalose. Mostpreferably, the saccharide is glucose. In an non-limiting exemplaryexample, a UCA, such as ST68, is diluted in 1:1 by volume with asolution of the saccharide. The final concentration of the saccharide inthe mixture with the UCA can be from about 1 millimolar (mM) to lessthan about 200 mM, preferably from about 10 mM to about 140 mM, and morepreferably about 50 mM to about 100 mM. In a preferred embodiment, thefinal concentration of the saccharide is about 90 to about 110 mM in themixture with the UCA. The mixture is then flash frozen, for instance, inliquid nitrogen, and then dried at a temperature of about −80° C. toabout −70° C. The container comprising the resulting freeze-dried UCA isthen purged, for instance with octafluoropropane gas, and the containersare sealed to prevent exposure of the lyophilized UCA to atmosphere.

UCA described herein can be reconstituted with any gas of choice,including, but not limited to, air, PFC or SF₆. An exemplary method offreeze drying and filling UCA with gas is as follows. An aliquot of aUCA suspension is placed in a 15 ml lyophilization vial (WestPharmaceutical Services, Lionville, Pa.). A Flurotec® lyophilizationstopper (West Pharmaceutical Services) is placed in the neck of the vialup to the first groove. The agent is then flash frozen in liquidnitrogen. The vial is placed on a previously chilled (initially to −80°C.) two-shelf stoppering rack of a Virtis Benchtop freeze-dryer(Gardiner, N.Y.) and freeze dried at pressures below 300 μBar and acondenser temperature of −76° C. For gas filling, the piston of thestoppering rack is lowered prior to venting the freeze dryer, thussealing the stopper in the vial under vacuum. The stoppered vial isremoved from the freeze dryer, and the gas of choice is introduced intothe individual vial from a tank via a needle pierced through the stopperseptum. A gas flow rate of 50 ml/min for the first 5 to 10 seconds andthen 20 ml/min for the next minute can be used to insure the vial isfilled. For capsules filled with air, the freeze drier is vented to theatmosphere prior to stoppering the vials.

As shown herein, the freeze-dried UCA advantageously is shelf stable forextended periods of time. Upon reconstitution, for instance withphosphate buffered saline, the reconstituted UCA retains a substantialdegree of echogenicity as compared to a non-lyophilized UCA. In someembodiments, the reconstituted UCA has at least about 50%, 55%, 60%,65%, 70%, or 75% of the enhancement exhibited by the same UCA that isfreshly prepared and not subjected to lyophilization. In someembodiments, the reconstituted UCA has at least about 80% of theenhancement exhibited by the same UCA that is freshly prepared and notsubjected to lyophilization. The reconstituted freeze-dried UCA of theinvention also possesses stability comparable to the same UCA that isfreshly prepared and not subjected to lyophilization.

In an embodiment of the method, the UCA is ST68, optionally particlestabilized, and lyoprotected with 100 mM glucose (ST68G-100). Exemplarymethods of making ST68 are known in the art (U. S. Pat. No. 5,352,436;Wheatley et al., 1995, Reactive Polymers, 25, 157-66; Wheatley et al.,2006, Ultrasound Med. Biol., 32, 83-93). Reconstitution of lyophilizedST68G-100 provides an UCA providing an in vitro peak US enhancement ofover 20 dB (37° C.; 5 MHz), with a half life of about 2.5 minutes andhaving measurable echogenicity for at least about 5 minutes andpreferably at least about 10 minutes.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Experimental Examples 1-4

The materials and methods employed in Examples 1-4 disclosed herein arenow described.

Surfactants

The surfactants for this study are three non-ionic surfactants, of whichtwo surfactants are fatty acid esters of sorbitan (SPAN and TWEEN) andthe third is water soluble vitamin E, (d-α-tocopheryl polyethyleneglycol 1000 succinate: TPGS). The potential compositions includecombinations of SPAN/TWEEN, SPAN/TPGS and SPAN/TWEEN/TPGS. The differentsurfactants in various combinations, in various concentrations, andencapsulating different gases (air, sulfur hexafluoride (SF₆) andperfluorocarbon, (PFC)) will dictate the packing density and strength ofinter-molecular forces around the gas. The agents will beoptimized/characterized in vitro for stability and echogenicity.

A mixture of two non-ionic surfactants, SPAN and TWEEN, was used tostabilize gas bubbles. Both surfactants are fatty acid esters ofsorbitan, which can have a hydrophobic tail group, for examplemonolaurate (C11: SPAN 20 and TWEEN 20), monopalmitate (C15: SPAN 40 andTWEEN 40), monostearate (C17: SPAN 60 and TWEEN 60), and monooleate(C17: SPAN 80 and TWEEN 80). All chains are fully saturated except forthe oleate chains which contain a single double-bond. The differencebetween TWEEN and SPAN is that TWEEN has the hydrophilic sorbitan headgroup modified with polyethyleneoxide groups (see FIG. 1) and thisgreatly increases polarity that makes the molecule more water-soluble.It should be noted that the stable microbubbles are successful only inthe combination between solid SPAN (SPAN 40 and SPAN 60) and almost alltypes of TWEEN. The trioleate series of SPAN 85 and TWEEN 85 do notstabilize bubbles in any combination, which is expected since theiroleate fatty acid chains are extremely bulky (Table 1; Wheatley et al.,1995, supra). Also, different combinations of surfactant and differentgases affect the backscatter from the microbubbles (Forsberg et al.,1999, Ultrasound in Med. And Biol. 25:1203-1211).

TABLE 1 Span 60 Span 40 Span 20 Span 80 Span 85 (solid) (solid) (liquid)(liquid) (liquid) 4.7^(a) 6.7 8.6 4.3 1.8 Tween 20 Y Y N N N (liquid)16.7^(a) Tween 40 Y Y N N N (liquid) 15.6 Tween 60 Y Y N N N (liquid)14.9 Tween 65 Y Y N N N (solid) 10.5 Tween 80 Y Y N N N (liquid) 15.0Tween 85 N N N N N (liquid) 11.0 Y: microbubbles were formed, N:microbubbles were not formed. ^(a)HLB values.

The previous model for microbubbles stabilized by SPAN 60 and TWEEN 80is shown in FIG. 2, and was developed using a Langmuir trough. The modelsuggested that the stability of the bubble is due to the fact that thebulky head of TWEEN is stabilized by the presence of SPAN in the shellwhich causes the reduction of the repulsive force in TWEEN molecules byhydrophobic attraction between the tail groups. This model, however,cannot explain why the microbubbles are stable only with solid SPAN.

Particle-Stabilized Foams

Particles can act as surfactants in stabilizing foams and emulsions(Binks, 2002, Curr. Opin. Colloid. Interface Sci. 7:21-41; Gonzenbach etal., 2006, Chem. Int. Ed. 45:3526-2530; Hunter et al., 2008, Adv.Colloid Int. Sci. 137:57-81). The solid particles can function in thesame ways as surfactants, but some behaviors are different. For example,particles do not always assemble the same way that surfactant moleculesdo when they form micelles, and, hence the solubilization phenomena(i.e. the ability of dilute surfactant solutions to solubilizedwater-insoluble substances to form stable systems; Harris, 1958, J. Am.Oil Chem. Soc. 35:428-435) is absent in the particle case. When thespherical particles adsorb to interfaces, the contact angle θ which theparticle makes with the interface is important (Binks, 2002, supra). Forhydrophilic particle, the contact angle measured into the aqueous phaseis normally less than 90° and the larger fraction of the particleresides in the water. By analogy with surfactants, the monolayer willcurve to make the larger area of the particle surface remain on theexternal side, giving rise to air or oil-in-water emulsions for θ<90°(FIG. 3). There will be an opposite effects for hydrophobic particles,which are suitable for water-in-air or water-in-oil emulsions with acontact angle which is greater than 90° (FIG. 3).

For the particle-stabilized foams, the stability is proportional toparticle concentration and inversely proportional to particle size. Thisis because smaller particles in high concentrations form a more completelayer thus giving the most effective steric barrier.

The physical reason for the better efficiency of particles oversurfactants in stabilizing foams is their attachment energy, which canbe up to several thousand kT per particle, where k is the Boltzmannconstant and T is the absolute temperature, compared to only a few kTper surfactant molecule (Dickinson et al., 2004, Langmuir 20:8517-8525).Because of this high energy attachment at the interface, the particleadsorption can be considered as irreversible (Vignes-Adler et al., 2008,Current Opinion in Colloids & and Interface Science 13:141-149. Therequired energy to remove the particle from its equilibrium position atthe interface to the bulk liquid phases is

ΔG _(remove) =πR ²σ(1±cos θ)²   (1)

where R is the radius of the spherical solid particle; σ is theinterfacial energy; θ is the contact angle; sign ‘+’ refers to particleremoval into gas phase, while sign ‘−’ refers to the removal into theliquid phase (Kaptay, 2006, Colloids and Surfaces A: Physicochem. Eng.Aspects 282-283:387-401). Equation 1, however, does not say anythingabout the stability of the thin liquid layer between bubbles which arestabilized by particles. To answer this question, the maximum capillarypressure was introduced and can be calculated from

ΔG _(remove) =πR ²σ(1⊥ cos θ)²   (2)

where p and z are the parameters for different particle arrangements(see FIG. 4). For example, in the case of closed-pack bilayer, if θ<90°,□□□p=4.27 and z=0.405 but for 90°≦θ<129.3°, p=2.73 and z=0.633. WithEquations 1 and 2, Kaptay can make the calculations that agree with theexperimentally observed optimum contact angle interval (Kaptay, 2006,ibid.).

Another approach to particle-stabilized foams is by changing thehydrophilicity and wetting properties of the solid particles so as tofavor their attachment at the gas-liquid interface. One possibility ismixing the colloid particles with amphiphilic molecules or surfactant.This scenario should fit well in our study using the particle of SPANmixed with TWEEN. The model and picture of this type of the system areshown in FIGS. 5 and 6.

Vitamin E TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate; TPGS)

TPGS is a water-soluble form of natural source vitamin E. It is verystable and does not hydrolyze under normal conditions. TPGS can beprepared by esterifying the acid group of d-alpha-tocopheryl acidsuccinate with polyethylene glycol 1000. The molecular structure of TPGSis shown in FIG. 8. It can be used as an emulsifier, drug solubilizer,absorption enhancer, and as a vehicle for lipid-base drug-deliveryformulations.

Nile Red

A hydrophobic dye, Nile Red, is a popular fluorescent probe inbiological and medical research used to localize and quantify lipids, tostain proteins, and to detect ligand-binding to enzymes. It is also usedas a florescent dye probe for the study of micelles (Maiti, et al.,1997, J. Phys. Chem. B 101:11051-11060; Krishna, 1999, J. Phys. Chem. A103:3589-3595; Nagy et al., 2003, J. Phys. Chem, A 107:8784-8790).Another interesting property of Nile Red is that its fluorescence isstrongly dependent on the polarity of its environment (Sackett et al.Wolff, 1987, Analytical Biochemistry 167:228-234). In DMSO, Nile red hasAbs/Em=552/636 nm. In cholesterol ester droplets or hydrocarbonsolvents, Nile red fluoresces yellow-gold (528 nm), while in ethanol orphosphatidylcholine vesicles, the dye fluoresces red (>610 nm; Greenspanet al., 1985, Journal of Lipid Research 26:781-789). In aqueous media,it is relatively insoluble and fluorescence is strongly quenched.

Type of Gases

Different types of gases including but not limited to, air, SF₆ and PFC,which have a different molecular weights, hydrophobicity, and watersolubility, may be used.

In Vivo Acoustic Testing

Dose response curves were generated in 3 kg New Zealand white rabbitswith 200 mM glucose stabilized freeze-dried ST68 (“ST68-g”). Each rabbitwas sedated with 35 mg/kg ketamine and 3.5 mg/kg xylazine. Increasingvolumes from 0.005, 0.01, 0.05, 0.1, to 0,15 mL/kg were injected throughan angiography catheter inserted in the left ear vein, followed by aflush with 5 ml of sterile saline. A total of 3 rabbits were used.Roughly 5 to 10 minutes passed between each dose to ensure totaldestruction of the agent and return to baseline values. A Sonix RP(Ultrasonix, Richmond, BC, Canada) scanner recorded all data received bythe L14-5 linear array set to 5 MHz with a PRF of 6.7 kHz and a gain of44% on pulse Doppler mode. This transducer was focused on themidabdominal aorta. Pulse inversion, with a power of −8 dB and a PRF of1 kHz, was used to image the kidney with a dose of 0.1 mL/kg. Thesestudies were carried out under the guidance of a veterinarian and allprotocols were approved by Jefferson University's Animal Care and UseCommittee. A similar dose curve was previously generated withfreshly-prepared ST68.

Size Characterization

All size measurements were carried out using a Zetasizer nano ZS(Malvern Inst, Worcestershire, UK). Twenty-five μL of agent wasdispersed into 975 μL of PBS and gently inverted to ensure thoroughmixing. For each sample, three measurements were taken and averagedtogether. The recorded value is the average±SEM (standard error of themean) of the z-average value.

Morphological Examination

ST68g and ST68 (freeze-dried without lyoprotectant) were prepared on analuminum specimen mount having excess blown off with pressurized air.Samples were carbon coated for 8 seconds using a Cressington 208bench-top carbon evaporator (Watford, England). Images were taken with aZeiss Supra 50 scanning electron microscope (S.E.M.) with Oxford EnergyDispersive Microanalysis (EDS) (Minnesota, USA) set to 3.5 kV and anaperture of 4 mm.

Statistical Analysis

All data is presented as mean±SEM with all experiments repeated at least3 times (n=3). For all data, statistical significance was determinedusing a one way ANOVA with a Newman-Keuls post test assuming normaldistribution and focusing in on comparisons with controls. All testingwas preformed using Prism 3.0 (GraphPad, San Diego, Calif.) using aprobability value cut off of 0.05 to determine statistical significance.

The results of Experimental Examples 1-4 are now described,

Experimental Example 1 Particle Stabilized Microbubble Foams asUltrasound Contrast Agents

It was observed that stable microbubbles can be formed only with a solidSPAN, i.e, SPAN 40 and SPAN 60, mixing with TWEEN. The mixture of SPAN20, a liquid form, with TWEEN or TWEEN alone did not give stablemicrobubbles, which agrees with the study of Wheatley and Singhal, 1995,Reactive Polymers 25:157-166. For this reason, it is possible that thestability of gas bubbles can be modeled as solid particle-stabilizedbubbles not a mixture of free surfactants.

The foam can be prepared by adding the desired amount of surfactant(s)to 25 ml phosphate buffered saline (PBS). The solution is then stirredand heated for 3-5 min until boiling or until the surfactant(s) iscompletely dissolved. The solution is then sterilized for 35 min atwhich point it is cooled to room temperature. To generate the foam, the15 ml of the solution is sonicated at maximum power for 3 min. The 20 mlof foam is pour into 25 ml graduated cylinder and amount of foam volumeis measure at various times.

After autoclaving and cooling down to room temperature, the solutions ofpure TWEEN and of low molar ratio of SPAN and TWEEN mixtures (i.e. 10:90and 20:80) were clear compared with solution using higher ratios (e.g.,higher than 30:80), which had a milky appearance. Size analysis of thepure TWEEN and a low ratio of SPAN to TWEEN solutions showed the samesize distribution (mean diameter of between 8-10 nm), which is in themicelle range, compared with the mean diameter of higher ratios of SPANto TWEEN that produced particles with more than 100 nm diameter. In thecase of low SPAN to TWEEN ratios, without wishing to be bound by theory,it is possible that the TWEEN in the mixture might increase thesolubility of SPAN by formation of mixed micelles. This interpretationagrees with a study by Eiser et al., (2007, Chem. Eng. Sci.62:1974-1987), which shows increasing solubility of a hydrophobiccomponent (e.g., fat particles) in the presence of a water-solublesurfactant. In the case of the high SPAN to TWEEN ratios, there is notenough TWEEN to form mixed micelles with SPAN, and TWEEN adsorbs ontothe SPAN particles, stabilizing the SPAN particles while the solutioncools down to room temperature after the sterilization. In this way,small particles of SPAN are formed by preventing the aggregation of SPANparticles, and the solutions appear cloudy.

Pictures were taken at periods up to 12 hours after foam was produced bysonicating solutions of pure TWEEN 80 (i.e. the pure, singlesurfactant-stabilized foams) and the mixture of SPAN 60 and TWEEN 80 inthe molar ratio of 80:20 (i.e. the particle-stabilized foams). It wasobserved that the surfactant stabilized-foam of pure TWEEN 80 is lessstable compared with the particle-stabilized foam of the mixture of80:20 of SPAN 60 to TWEEN 80. For the surfactant-stabilized foam (i.e.pure TWEEN 80 and the mixture of SPAN 60 and TWEEN 80 at molar ratio of20:80), the foam volume reduces to less than 10% of initial volumeexponentially, within 12 hours. In the case of particle-stabilized foam(i.e. the mixture of SPAN 60 and TWEEN 80 at molar ratio of 80:20), thefoam is very stable, dropping to 37.5% of initial volume and remainingat that volume for even more than 3 days.

Without wishing to be bound by any particular theory, it is hypothesizedthat particles of SPAN stabilize the bubbles. A possible model could bean adaptation of the models that were originally proposed and is shownin FIGS. 5 and 6. It is proposed that the TWEEN 80 molecules stabilizethe system by adsorbing around and between the hydrophobic SPAN 60particles and the solution while part of the SPAN 60 particles should beadditionally stabilized while in contact with the hydrophobic gas (FIG.9).

A Standard Procedure Producing Microbubbles

To fabricate the microbubbles, the desired amount of SPAN with TWEEN,and, optionally, sodium chloride are added into 50 ml PBS. The solutionis then stirred and heated for 3-5 min, until boiling or until SPAN isdissolved. The solution is then sterilized for 35 minutes after which itis cooled to room temperature. To generate the bubbles, the cooledsolution (held in an ice bath) is sonicated at 110 W for 3 min in thepresence of the desired gas to be entrapped. The bubbles are then washedthree times with 50 ml PBS in a separating funnel. The solution isallowed to separate into three distinct layers (about 35 minutes) andthe bottom layer consisting of unused surfactants is discarded with eachwash. After the last wash, the microbubbles at the middle layer arecollected.

The microbubbles are named after the mixture of SPAN and TWEEN that isused. For example, ST44 consists of SPAN 40 and TWEEN 40, ST48 consistof SPAN 40 and TWEEN 80 and ST68 consist of SPAN 60 and TWEEN 80. One ofthe beauties of the agent is the range of combinations that is possible.

Acoustic Testing

The acoustic properties of microbubbles were tested in vitro by anacoustic set up. A one-dimensional pulsed A-mode US set-up with a singleelement, broadband, 12.7 mm element diameter, 50.8 mm sphericallyfocused transducers with center frequencies 5 MHz (Panametrics, Inc.,Waltham, Mass.). The −6 dB bandwidths of the transducers were 89%, 92%,71% and 65%, respectively. The transducers were inserted in a water bathfilled with deionized water, (37° C.) and focused through an acousticwindow of a 100 ml custom-made sample vessel, A pulser/receiver (model5072 PR, Panametrics, Inc., Waltham, Mass.) was used to pulse thetransducers at a pulse repetition frequency (PRF) of 100 Hz. Thereceived signals were amplified to 40 dB and fed to the digitaloscilloscope (Lecroy 9350A, Lecroy, Chestnut Ridge, N.Y.). The digitizeddata was stored and analyzed using Labview (National Instruments,Austin, Tex., USA). The bubbles were injected into the sample chamberusing an automatic pipette, and stirred with a magnetic stirrerthroughout the readings. The reference (PBS) is taken as an average ofsix values. Readings with buffer alone indicate that this method doesnot introduce unwanted air bubbles into the sample chamber. Enhancementand attenuation were calculated as a function of dose and time.

There are two curves which can be constructed from the acoustic set up.One curce is a dose response curve that demonstrates the echogenicity(dB of impinging sound that is reflected back to the transducer) of thebubbles. For each dose, a sample of the bubbles was added into 50 mL ofPBS in the custom-made vessel then calculated and reported in the unitof μL of the bubbles per liter of PBS (μL/L). All of the dose responseresults are not cumulative. Another curve is a time response curve thatdemonstrates the testing of the stability of the bubbles overtime, underconstant insonation. A dose on the linear rise of dose response must bechosen to conduct an accurate time response curve.

Particle Stabilization Theory Tested with Microbubbles Stabilized byMixed SPAN and TWEEN

FIG. 10 shows size distribution and the time response curves ofmicrobubbles stabilized by mixing 2 m mole of SPAN 60 with variousconcentration of TWEEN 80 (0.1, 0.5, and 1 m mole) in 50 ml of PBS with1.5 g NaCl. All are above the CMC of TWEEN 80, which is 0.012 mM. Fromthe size distribution results (FIG. 10A) one can see that the meandiameter of the particles decreases as the amount of TWEEN 80 increasesrelative to a constant amount of SPAN 60. When testing the bubblesproduced from these mixtures in vitro, the bubbles were more stable asthe diameter of the particles increased. The half life of themicrobubbles (t_(1/2)) is 6.02±0.48, 7.41±0.67, and 18.42±0.48 min. for1, 0.5, and 0.1 m mole of TWEEN 80, respectively (FIG. 10B). Theseresults agree with equation (1) that the bigger the particles, the moreenergy is needed to remove the particles from the interface. It shouldbe note that the microbubbles produced from 2 m mole of SPAN 60 with 0.5and 0.1 m mole of TWEEN 80 are statistically significantly more stablecompared with standard ST68 (mixture of 3.44 m mole of SPAN 60 and 0.81m mole of TWEEN 80, t1/2=5.20±0.20 min.), p=0.034 and 1.82×10-4,respectively. These results suggest that the behavior of microbubbles inin vitro testing can be explained by the particle stabilization theory.

Experimental Example 2 The Method for Fabricating Nano-Sized ContrastAgent

The Nanobubbles Produced from Higher Sonication Power

The mixture of nano- and micro-sized bubbles produced by the standardprocedure can be separated by centrifugation (Oeffinger et al., 2004,Ultrasonic 42:343-347). One method to increase the proportion thenano-size bubbles is to change the sonication power during fabrication.Sonication at the power of 140 W instead of 110 W yield morenanobubbles, depending on the sonication time. For the sonication timeof 90 and 120 sec., including 3 min, there are three distinct layers,which is the same as observed in the standard procedure to produce ST68,The size analysis of the collected middle layer is also in the range ofstandard microbubbles, which are in between 1-2 μm. It is different forthe 30 and 60 sec. sonication time in that there is no distinct layer at35 min. after washing step. If we assume that this lack of developmentof a distinct middle layer is due to the fact that there is a largepopulation of very slowly rising nanobubbles, we can hasten the processand instead of waiting for these to rise and form a layer, we candiscard the lower 20 ml of solution that contains unused surfactant, andcollect the next 25 ml, which is the nano-rich layer. The mean diameterof the bubbles in this layer is around 787 nm. The echogenicity testingof this sample also shows that these “nanobubbles” gives almost the sameas standard ST68 except that a higher dose is required to get the sameresult as standard ST68. From this result, it is possible that theamount of the nanobubbles can be increased by modifying some parametersand the protocol to collect the bubble of the current procedure to getthe nanobubbles.

Nanobubbles Produced by Changing Surfactant Components

Another potential method to produce a larger proportion of nanobubblesis to change the surfactant composition. The standard procedure wascarried out except for the washing and collecting step. Because therewas no distinct layer after one hour after the first wash with 50 mlPBS, the lower 25 ml was discarded and then the next 50-75 ml of themixture was transfer to a separation funnel B while the remainingmixture was left in funnel A. Both separation funnels A and B are thencarried through the washing and separation step in parallel, beingwashed with 50 ml PBS twice. The Funnel A produced three distinct layersafter 35 min. standing after both washing step. The middle layer wascollected in the same procedure as for standard ST68 and these bubblesare called SE61. SE61 had the same mean diameter in the range ofstandard ST68, which is in between 1-2 μm. For the newly transfer funnelB, each washing step was left to separate for one hour. At the end ofthe waiting period, the lower 20 ml of solution was discarded from theseparating funnel. The mixtures was collected after the last discard andcalled nSE61. These nanobubbles have mean diameter of 337.67±19.77 nm.

The normalized time response of SE61 and nSE61 compared with standardST68 show that the differences among all three agents are statisticallysignificant (p=0.0485 for ST68 and SE61 and p=3.72×10-4 for ST68 andnSE61). The SE61 (t½=10.18±1.77 min.) is more stable than the standardST68, which will be a benefit for using it as a contrast agent. However,nSE61 (t½=1.75±0.24 min.) is considerably less stable than standardST68. A susceptibility to ultrasound of the nano agent might be abenefit for the drug release when using the nanobubbles as a drugcarrier.

Experimental Example 3 Drug Delivery

The agent ST68 was chosen for the preliminary study of Nile Red loadedmicrobubbles, since this agent is well characterized. Nile Red was addedinto the solution after the sterilization step and then heated until thesolution boiled for 3-5 min. At this point, the solution color changedfrom milky white to pink or red depending on the amount of Nile Red thatwas added. This is due to Nile Red becoming dissolve into micelles, andfluorescing in the nonpolar environment of the micelle. The solution wasthen left to cool down to room temperature. Microbubbles were then madeby the usual protocol (sonicate at 110 W for 3 min.).

The qualitative analysis of Nile Red intercalated to the bubbles can betested by the fluorescent property of Nile Red. It is strongly quenchedin aqueous media but fluoresces depending on the polarity of itsenvironment. As Nile Red is intercalated into the hydrophobic shell ofthe bubbles, under fluorescent microscopy, the shell of the bubblesfluoresce as can be seen.

The echogenicity of Nile Red loaded microbubbles was tested. It wasfound that both the dose and the time response of the Nile Red loadedbubbles are not statistically significantly different (p<0.05) fromregular ST68.

Micelles of Low Ratio SPAN 60 to TWEEN 80 for Drug Delivery

Another approach to nanocarriers for poorly water-soluble drug deliveryis to use polymeric micelles. The hydrophobic drugs can be solubilizedin their hydrophobic inner cores. This study of using a mixed surfactantsystem for a contrast agent also makes possible a study of the mixedmicelles of these surfactants as drug carriers. At the ratio of 20:80,the mixed micelle of SPAN 60 and TWEEN80 can be formed without suspendedparticles and the mean size of these micelles is around 9.9 nm.

The qualitative analysis for solubilized Nile Red was analyzed by HPLCwith acetonitrile as mobile phase with UV detector at λ_(max) 538 nm.This wavelength was found to be correct for the maximum absorbance ofNile Red dissolved in acetonitrile using the Plate Reader. A calibrationcurve of Nile Red in acetonitrile analyzed by HPLC was established(R²=0.9999). It was discovered that SPAN and TWEEN interfered with theanalysis of Nile Red. As a modification, petroleum ether was first usedto extract Nile Red from the mixture by mixing 5 mL of the mixture with5 mL of Petroleum Ether. This mixture was shaken for 15 sec., and thenplaced in the centrifuge with a setting of 9000 rpm (4500 g) for 30 min.After the centrifugation, 2 mL of the top layer of petroleum ether waspipetted into a glass vial, and the petroleum ether was allowed toevaporate under the fume hood. Two mL of acetonitrile was added todissolve Nile Red. To make sure that Nile Red completely dissolved, thevial was sealed and shaken for 48 hr before being analyzed by HPLC. Fromthe results, one can see that in the solution of total concentrationsurfactant of 85.1 mM, Nile Red can be solubilized in the mixed micellesat the maximum concentration of 47 mg of Nile Red in one liter ofsolution.

The echogenicity of the mixtures of the bubbles and the micelles isaround 25 dB at a dose of 50 μl/L and t_(1/2) around 5 min. This testshows that the equilibrium of the bubbles system is not altered bymixing in the micelles, at least in 30 minutes, and makes possible themixing system for drug delivery.

Experimental Example 4 Freeze-Drying Enhances Storage and Stability ofthe UCA

To assess lyoprotection, four candidate saccharides were tested aslyoprotectants: glucose, trehelose, sucrose and mannitol.

Aliquots of 2 mL undiluted ST68 are placed in 15 mL lyophilization vials(West Pharmaceutical Services, Lionville, Pa.) in a 1:1 volume ratio of200 mM (1.8 w/v %) sugar lyoprotectant dissolved in 18.6 MΩ cm deionized(DI) water. Samples were frozen in liquid nitrogen while FLUORTEK®lyophilization stoppers (West Pharmaceutical Services) were placed onthe vials to the first groove. The cryoprotected ST68 was dried on apreviously chilled (−80° C.) shelf for 20 to 24 hours using a VirtisBenchtop freeze-dryer (Gardiner, N.Y.) at pressures below 200 μBar.Prior to venting, a piston was lowered thus sealing the stoppers on thevials. After the vials were shaken and tapped on the table to dispersethe particles from their freeze-dried cake, octafluoropropane gas wasintroduced, via needle, into the vials through the stopper septum at aflow rate of 6 mL/min for the first 5 to 10 seconds, then 4 mL/min for3½ minutes to insure the vials were filled. Filling time was adjustedbased on the volume (15 mL) of the lyophilization vials. Parafilm waswrapped around the stopper/vial seal to prevent gas diffusion. Beforeuse, the freeze-dried ST68 was reconstituted with 1 mL DI water and 1 mLphosphate buffered saline (PBS) both at 4° C. by hand agitating.

Effect of Choice of Lyoprotectant on UCA Size

For all lyoprotectants, bubble size remained constant at 3±0.15 μm. Allsamples were significantly less than 6 μm, ensuring they would be ableto transverse the pulmonary capillary bed.

In Vitro Acoustic Performance

Both glucose and trehalose provided statistically greater in vitroenhancement (p<0.001) than that of the reconstituted ST68 control(freeze-dried without the addition of any lyoprotectant), both giving apeak enhancement of about 23 dB, comparable to the enhancement offreshly prepared ST68. Samples lyophilized in the presence of sucroseand mannitol provided a peak enhancement of about 18 and 19 dB,respectively, but were not statistically greater than the control, whichyielded about a 17.5 dB peak enhancement. For all sugar controls(freeze-dried dissolved sugar in PBS without the addition of ST68), amaximum enhancement of about 2 dB was recorded signifying that thelyoprotectant itself did not cause the increase in enhancement over thatof the ST68 samples.

A study of stability was also completed for all lyoprotectants at roomtemperature (20° C.) and body temperature (37° C.) for comparison. At37° C., there was no statistical different between the half lives of anyreconstituted ST68 with or without lyoprotectants. However, after thefirst rapid loss of echogenicity, the samples preserved with glucoseretained the highest residual activity (20% at 15 minutes compared to˜5% for freshly prepared agent).

However, glucose provided longer stability at 20° C. over the ST68control (p<0.01) and all other lyoprotectants (p<0.05 over sucrose and<0.01 over all others). Reconstituted ST68 even at 37° C. retainsmeasurable echogenicity for over 10 minutes in vitro.

Effects of Freeze-Drying and Lyoprotectants on Reconstitution

Upon reconstitution, mannitol, sucrose, and control samples were unableto be completely re-suspended, leaving behind visually largeparticulates which might prove dangerous if injected into the body.These larger particles evaded detection in the size analysis due totheir buoyancy.

Glucose Concentration Optimization

A range of glucose concentrations, from 20 mM to 400 mM or 0.2 to 3.6w/v %, was tested for optimization. While not statistically significant,200 mM of glucose (ST68G-200) provided a 4 dB greater enhancement overthe other concentrations, providing a peak enhancement of about 23 dB.However, the half life at 37° C. of all glucose cryoprotected agentsremained constant at an average of 3+0.3 minutes signifying theconcentration of glucose did not significantly affect the ST68stability.

In Vivo Study

The in vivo dose response experiments of ST68G-200 were modeled afterprevious studies (Forsberg et al., 1996, supra; Wheatley et al., 2006,supra) of freshly prepared ST68 for direct comparison. An average peakenhancement of about 23 to about 25 dB were recorded for the freshlyprepared ST68. The freeze-dried agent ST68G-200 was chosen for thisstudy based on the results outlined above, and provided a peakenhancement of about 21 dB, being on average 3 dB under that of thefreshly prepared ST68.

Pulse inversion harmonic imaging (5 MHz) of a New Zealand white rabbitkidney pre and post injection of ST68G-200 was performed. Thevasculature and parenchyma boundaries of the kidney are clearly visibleafter injection of 0.1 mL/kg contrast.

Microscope Imaging

To show the difference between ST68g-200 and ST68 without lyoprotectant,S.E.M. images were taken of both. Protected bubbles can be seen in withlyoprotectant present while without addition of a lyoprotectant, bubblesare not present.

Experimental Examples 5-11

Experimental Examples 5-11 are directed to describing and characterizingin more detail the lyoprotection of the UCA studied in ExperimentalExample 4. The materials and methods for Experimental Examples 5-11 arenow described.

Materials

SPAN 60 (sorbitan monostearate), TWEEN 80 (polyoxyethylene sorbitanmonooleate), potassium chloride, sucrose, D-glucose anhydrous,D-mannitol, D-trehalose dihydrate, potassium phosphate monobasic, sodiumchloride, and sodium phosphate dibasic were all purchased fromSigma-Aldrich (St. Louis, Mo.). Octafluropropane (99% min) was purchasedfrom American Gas Group (Toledo, Ohio).

Sample Preparation

ST68 was manufactured using a method developed in our laboratory(Wheatley et al., 1994; Wheatley and Singhal 1995; Wheatley et. al2006). Aliquots of 2 ml of non3 diluted ST68, were placed in 15 mllyophilization vials (West Pharmaceutical Services, Lionville, Pa.) anddiluted with 2 ml of a solution of selected sugar lyoprotectantdissolved in DI water. Samples were flash frozen in liquid nitrogen(shown to prevent sample and solvent separation (Costantino and Pikal2004) and improve the redispersion of nanoparticles (Lee et al., 2009)with FLUOROTEK® 7 lyophilization stoppers (West Pharmaceutical Services)placed on the vials to the first groove as exemplified by Jennings(1999). The lyoprotected ST68 was dried on a previously chilled(initially to −80° C.) shelf for 18 to 20 hours using a Virtis Benchtopfreeze-dryer (Gardiner, N.Y.) at pressures below 300 μBar and acondenser temperature of −76° C. Prior to venting, a piston was loweredthus sealing the stoppers on the vials. To measure the temperatureprofile of the samples, a thermocouple was frozen within the center ofthe matrix prior to lyophilization. The temperature of the frozen matrixwas recorded every 10 minutes on a remote 4 Channel DataloggerThermometer (Spec Scientific LTD, Scottsdale, Ariz.) for the duration ofthe freeze-drying process.

After the ST68 samples were lyophilized, octafluoropropane gas wasintroduced via a needle into the vials through the stopper septum at aflow rate of 50 ml/min for the first 5 to 10 seconds then 20 ml/min forthe next minute to insure the vials were filled. Finally, parafilm waswrapped around the stopper/vial seal to prevent gas diffusion. Beforeuse, the freeze-dried ST68 was reconstituted by hand agitation with 2 mlDI water and 2 ml phosphate buffered saline (PBS), both at 4° C.,yielding a 1:1 dilution facto compared to the original non-dilutedsample.

Residual Water Content

Freeze-dried samples of ST68 in lyophilization vials were unstopperedand weighed. Each vial was then placed within an Imperial III incubator(Lab-Line Instruments Inc., Melrose Park, Ill.) set to 60° C. for 24hours and weighed again. This procedure was repeated until the weight ofthe samples remained constant indicating that all residual water hadbeen removed. The water content was calculated as a percentage ofinitial weight.

Size Characterization

All size measurements were carried out using a Zetasizer nano ZS(Malvern Inst., Worcestershire, UK). Twenty-five μl of agent wasdispersed into 975 μl of PBS and gently inverted to ensure thoroughmixing. For each sample, three measurements (z13 average diameter whichwas found to be more consistent in measuring ST68 size than number orsize average) were taken and averaged together.

Microscope Imaging

Polarized Light Microscopy (PLM) images of ST68 samples were taken ofindividual drops of ST68 with each excipient. These samples werepreviously placed onto a glass slide and frozen in a −80° C. freezerbefore being lyophilized overnight, PLM images were taken at 20× with anOlympus BX50 model U-SDO (Tokyo, Japan) using PixeLINK Capture OEM 2005software (Ottawa, ON, Canada).

Samples of ST68G-100 (100 mM glucose stabilized freeze-dried ST68) andST68 control (freeze-dried without lyoprotectant) were prepared on analuminum specimen mount, previously covered with a 12 mm non1-conductive adhesive tab, having excess sample blown off withpressurized air. Samples were then carbon coated for 8 seconds using aCressington 208 bench-top carbon evaporator (Watford, England). Imageswere taken with a Zeiss Supra 50 (Cambridge, Cambridgeshire, UK)scanning electron microscope (S.E.M.) with Oxford Energy DispersiveMicroanalysis (EDS) (Abingdon, Oxfordshire, UK) set to 3.5 kV and withan aperture of 4 mm.

In Vitro Acoustic Setup

An acrylic sampling container holding 50 ml of 37° C. PBS, housing anacoustic viewing window of 3×3 cm, was placed within a larger acrylictank holding 20 gallons of 37° C. DI water to be used for acoustictesting of the samples, as previously described (Basude et al., 2000).The contents of the sampling container were continuously stirred with amagnetic stirrer between 200 and 400 rpm. A Panametrics (Waltham, Mass.)5 MHz transducer with a 12.7 mm diameter, −6 dB bandwidth of 91%, andfocal length of 50.8 mm was focused through the sampling window.Acoustic pressure amplitudes were generated with a Panametricspulse/receiver (5072 PR) at a pulse repetition frequency (PRF) equal to100 Hz. Using a 0.5 mm polyvinylidene fluoride needle hydrophone(Precision Acoustics, Dorset, UK), peak positive and negative pressureswere measured at 0.69 and 0.45 MPa, respectively. Received signals wereamplified 40 dB and read using a digital oscilloscope (LeCroy 9350A,LeCroy Corporation, Chestnut Ridge, N.Y.). Labview 7.1 express (NationalInstruments, Austin, Tex.) was utilized to process the data.

In Vitro Dose and Time Response

Quantities of test samples of ST68 were measured by pipette (GilsonPipetman, Middleton, Wis.) and dispersed into the sampling container. Acumulated dose curve (expedient for discerning comparisons betweenlyoprotectants) was generated by pipetting increments of agent into thesample chamber while measuring the acoustic response. The curve was usedto determine the dose at which maximum reflection was achieved and toassess differences between samples prepared with various lyoprotectants.Shadowing occurred when the concentration of bubbles was high, thuscausing a total reflection of the US signal (Bogdahn et al., 2001,Transcranial color-coded duplex sonography (TCCS). In: Dunitz, M. (Ed.)Ultrasound Contrast Agents: Basic principles and clinical applications.Martin Dunitz Ltd., London, UK., pp. 253-65). To examine the stabilityof the UCA while being exposed to an ultrasound beam, samples on therise of the dose response curve (100 μl/l for reconstituted freeze-driedsamples and 30 μl/I for the native agent) were insonated over a 15minute period with readings taken every minute, after a 10 second delayto allow for sample mixing. The chosen volumes gave similarconcentrations of microbubbles (2.5 to 3.0×1.0⁹microbubbles permilliliter as measured by a hemocytometer) and were selected to preventanomalous high stability readings that would be obtained by recordingunchanged enhancement from degrading bubbles in an over-saturatedsystem. Data was normalized by the initial dB value to allow for intersample comparison. Half-life data was extracted from the response of theagents over time by fitting a line to the section of the curve whichpassed through 50% of maximum enhancement. For all, a native ST68 and afreeze-dried (not lyoprotected) control were used for comparison.

In Vivo Acoustic Testing

Dose response curves were generated in three 3 kg New Zealand whiterabbits with ST68G-100. Each rabbit was sedated with 35 mg/kg ketamineand 3.5 mg/kg xylazine. Increasing volumes, from 0.005, 0.01, 0.05, 0.1,to 0.15 ml/kg were injected through an angiography catheter insertedinto the left ear vein, followed by a flush of 5 ml sterile saline.Roughly 5 to 10 minutes passed between each dose to ensure total removalof the agent and a return to baseline values. A Sonix RP scanner(Ultrasonix Medical Corp., Richmond, BC, Canada) recorded all datareceived by the L14-5 linear array set to 5 MHz with a PRF of 6.7 kHzand a gain of 44% in pulse Doppler mode, having been focused on the midabdominal aorta. Pulse inversion harmonic imaging (PIHI), with a powerof −8 dB and a PRF of 1 kHz, was used to image the kidney with a dose of0.1 ml/kg. These studies were carried out under the guidance of aveterinarian and all protocols were approved by Jefferson University'sAnimal Care and Use Committee. A similar dose curve was previouslygenerated with native and nano ST68 (Forsberg et al., 1996, supra;Wheatley et al., 2006, supra).

Statistical Analysis

All data is presented as mean+SEM (standard error about the mean) withall experiments repeated at least 3 times (n=3). For all data,statistical significance was determined using a one-way ANOVA with aNewman-Keuls post test assuming normal distribution and focusing oncomparisons with controls. All testing was performed using Prism 3.0(GraphPad, San Diego, Calif.) with a probability value cut off of 0.05chosen to determine statistical significance.

The results for Experimental Examples 5-11 are now presented.

Experimental Example 5 Effect of Each Lyoprotectant on the UCA

For all four candidate lyoprotectants, bubble size remained constantwith an average of 3+0.2 μm with no statistical difference calculatedbetween samples (p>0.05). These results are larger than previouslyreported (Basude et al., 2000, supra). All samples were significantlyless than 8 μm, ensuring they would be able to transverse the pulmonarycapillary bed (Bouakaz et al., 2007, Ultrasound Med. Biol., 33, 187-96).

Upon visual inspection, it was apparent that large particles remainedafter reconstitution for the mannitol and sucrose samples; this alsooccurred with non-lyoprotected ST68 control samples. Since these largeparticles were buoyant, they rapidly rose to the top of the cuvette andthus eluded Zetasizer measurements which are based on Brownian motion ofparticles in the target area. The observation that theglucose-lyoprotected sample did not result in large particles isunexpected. Literature suggests that the particles freeze-dried in thepresent of glucose would also have reconstitution problems (Abdelwahedet al., 2006a, supra).

During lyophilization, the thermocouples embedded in sample vialsindicated that glucose was able to keep the sample at a temperature of−12° C. (zero slope, linear portion of the temperature/time curve; FIG.12) for the longest period of time (7 hours). Trehalose and sucrosemaintained this temperature for only 2 hours, while mannitol merelypassed through, exhibiting a constantly rising temperature profile. Allsamples, aside from mannitol-protected ST68, had an initial rise intemperature to the 2 hour mark followed by a cooling to the −12° C.steady state sublimation temperature.

After lyophilization, all of the samples had between 2 and 6% residualwater content (Table 2), with no statistical differences measured(p>0.05). Trehalose samples had the lowest (2.2+0.2%) while glucose andcontrol samples were around 5%. Water content alone did not affect theoverall echogenicity or stability of the sample.

TABLE 2 Excipient Water Content (%) Glucose 5.0 ± 0.2 Trehalose 2.2 ±0.2 Sucrose 4.2 ± 1.3 Mannitol 4.1 ± 0.1 ST68 (control; no saccharide)5.2 ± 0.8

Experimental Example 6 In Vitro Acoustic Performance

At 100 mM (Huang et al., 2002, supra), both glucose and trehaloseprovided statistically greater in vitro enhancement (p<0.001) than thatof the reconstituted sucrose, mannitol, and the ST68 control(freeze-dried without the addition of lyoprotectant) (FIG. 13). A peakenhancement of 23.2±1.2 dB and 21.9±0.7 dB were measured for glucose andtrehalose, respectively, both being statistically equivalent (p>0.5) tothe 24.5±0.2 dB enhancement of native ST68. Samples lyophilized in thepresence of sucrose and mannitol provided a peak enhancement of 17.9±0.1dB and 18.9±0.5 dB, respectively, but were not statistically greaterthan the control, which yielded a 17.6±1.6 dB peak enhancement (p>0.05).For all sugar controls (freeze-dried dissolved sugar in PBS without theaddition of ST68), an average enhancement of 0.4±0.1 dB was recorded,signifying that the lyoprotectant itself did not have any inherentechogenic properties.

Although all the tested lyoprotectants resulted in lyophilized materialthat reconstituted to give over 15 dB of enhancement, glucose andtrehalose provided the best protection resulting in a 5 dB increase overthe others. It is surprising that both glucose and trehalose providedthe best protection. It has been reported the protective effects ofsaccharides are proportional to their glass transition temperatures (Tg;Hua et al., 2003, supra). Experimental and calculated glass transitiontemperatures (T_(g)) of some of the pure sugars of interest have beenreported as trehalose (107° C.), sucrose (60° C.), glucose (23° C.)(Simperler et al., 2006, J. Phys. Chem. B, 110, 19678-84), whilemannitol is reported at 11° C. (Yu et al., 1998, J. Pharm. Sci., 87,774-7). The studies in Hua exhibited that the retention rates forreconstituted freeze-dried liposomal contents followed the same trend asthe T_(g), indicating that the best protection was from trehalose andthe worst from glucose. Therefore, the equal protective effects ofglucose and trehalose demonstrated here are unexpected and suggests thatthere is more involved in stabilizing UCA than with liposomes.

A study of stability (FIG. 14) was completed for all lyoprotectedsamples at room temperature (23° C.) and body temperature (37° C.). At23° C. glucose provided longer stability (12.1±0.6 min.) over the ST68control (5.8±0.8 min., p<0.01.) and all other lyoprotectants (p<0.05over sucrose and <0.01 over rest). After 15 minutes, samples preservedwith glucose retained the highest residual activity (45%; 7±0.4 dB) overall the other lyoprotected samples (p<0.05 for sucrose and ST68 control,p<0.01 for trehalose and mannitol) and did not statistically differ fromnative ST68 (55%; 11.7±0.9 dB) over the duration (FIG. 15A). At 37° C.,there were no statistical differences (p>0.05) between the half lives ofany reconstituted lyoprotected ST68 when compared to the nakedreconstituted control, yielding an average half life of 3.1±0.5 minutes(FIG. 14) and sustaining measurable echogenicity for over 10 minutes(FIG. 15B).

Experimental Example 7 Polarized Light Microscopy

PLM images were taken to better describe the apparent mechanism ofstabilization of the different sugars. The images illustrate thatmannitol (FIG. 16D) is the only excipient that crystallized whileglucose (FIG. 15A), trehalose (FIG. 15B) and sucrose (FIG. 15C) formedamorphous glassy matrices. ST68 control, without any lyoprotectant (FIG.15E) is amorphous as well, but does not show any presence of a glassymatrix.

Experimental Example 8 Glucose Concentration Optimization

A range of final glucose concentrations, from 10 mM to 200 mM or 0.2 to3.6% w/v, was tested for optimization. This range is consistent withconcentrations used for freeze-drying in literature (Jeong et al., 2005,J. Microencap., 22, 593-601). While not statistically significant(p>0.05), 100 mM of glucose-protected samples (ST68G-100), 1.8% w/v,provided a 4 dB greater peak enhancement (23.2±1.2 dB) over the otherconcentrations (FIG. 17). The half life at 37° C. of all glucoselyoprotected agents, however, remained constant at an average of 2.8±0.1minutes, signifying that the concentration of glucose did notsignificantly affect the stability of ST68 (FIG. 18). Yet, with 200 mMof glucose, the final product (cake) after freeze-drying had evidence ofcollapse, melt back (thawing during drying), and crystallization. Thiscaused reconstitution difficulties.

Experimental Example 9 Shelf-Life Study

ST68G-100 was tested at 100 μl/l for stability at the start of eachmonth over a period of 3 months. No statistical differences were found(p>0.05), having an average maximum enhancement of 19.6±1.0 dB and ahalf-life of 2.6±0.1 minutes for the duration (FIG. 19). Originally,ST68 would be stable for a maximum of a few weeks, being stored at 4°C., before the collapse and coalescence of bubbles decreased the effectof the agent. Having a freeze-dried form of this agent stable for over 3months at room temperature negates the need for immediate productionprior to use.

Experimental Example 10 Scanning Electron Microscope Imaging

To show the difference between ST68G-100 and ST68 control (withoutlyoprotectant), S.E.M. images were taken of both samples afterfreeze-drying and PFC gas introduction. Protected bubbles can be seen inFIG. 20A while, without the addition of a lyoprotectant, bubbles are notpresent (FIG. 20B). The ruptured capsule in FIG. 20A clearly shows thehollow nature of these particles, as well as their fragile nature, sinceit could have been ruptured during sample preparation for imaging.

Experimental Example 11 In Vivo Study

The in vivo dose response experiments of ST68G-100 (FIG. 10) weremodeled after previous studies (Forsberg et al., 1996, supra; Wheatleyet al., 2006, supra) of ST68 for direct comparison. Previously, amaximum peak enhancement of 26.1±0.5 dB (Forsberg et al., 1996, supra)and 23.7±2.9 dB (Wheatley et al., 2006, supra) were recorded for thenative and nano ST68, respectively. The lyophilized agent, ST68G-100,was chosen for this study based on results outlined above and provided apeak enhancement of 20.8±0.8 dB, being 4 dB below the recorded averageof native and nano ST68, A PUTT (5 MHz) of a New Zealand white rabbitkidney pre- and post-injection of a reconstituted freeze-dried ST68G-100sample is depicted in FIG. 11. The vasculature and parenchyma boundariesof the kidney are clearly visible after injection of 0.1 ml/kg contrast.Additionally, pulse Doppler images (results not shown) of the same agentand injection volume were comparable to non-freeze dried examples.

These data demonstrate that surfactant-stabilized gas bubbles, such asST68, can be freeze-dried, stored at about 4° for extended periods oftime, and reconstituted, while successfully maintaining echogenicityboth in vitro and in viva and stability (e.g., half life).

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A freeze-dried ultrasound contrast agent (UCA) comprising at least afirst surfactant, a second surfactant and a saccharide.
 2. Thefreeze-dried UCA of claim 2, wherein the saccharide is selected from thegroup consisting of glucose and trehalose.
 3. A reconstituted UCA,comprising a freeze-dried UCA and an excipient, wherein the freeze-driedUCA comprises at least a first surfactant, a second surfactant and asaccharide.
 4. The UCA of claim 1, wherein the surfactant is selectedfrom the group consisting of SPAN, alkylphenol ethoxylate-basedsurfactants, alcohol ethoxylate-based surfactants, silicone-basedsurfactants, alkyl poly(ethylene oxide), alkylphenol poly(ethyleneoxide), copolymers of poly(ethylene oxide) and poly(propylene oxide),alkyl polyglucosides, fatty alcohols, cocamide MEA, cocamide DEA, andpolysorbates.
 5. The UCA of claim 1, wherein the UCA is aparticle-stabilized UCA.
 6. The UCA of claim 1, wherein the firstsurfactant is TPGS.
 7. A particle-stabilized UCA comprising at least afirst surfactant and a second surfactant, wherein the first surfactantis d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), whereinsaid UCA further comprises a particulate material that stabilizes theUCA, and wherein the diameter of said UCA is between 1 nm and 1 μm. 8.The UCA of claim 7, wherein the second surfactant is selected from thegroup consisting of SPAN, alkylphenol ethoxylate-based surfactants,alcohol ethoxylate-based surfactants, silicone-based surfactants, alkylpoly(ethylene oxide), alkylphenol poly(ethylene oxide), copolymers ofpoly(ethylene oxide) and poly(propylene oxide), alkyl polyglucosides,fatty alcohols, cocamide MEA, cocamide DEA, and polysorbates.
 9. Amethod of making a lyoprotected ultrasound contrast agent (UCA), saidmethod comprising the steps of: a) preparing a UCA comprising at least afirst surfactant and a second surfactant; and b) adding a lyoprotectantto the UCA to prepare a lyoprotected UCA, wherein the lyoprotectant is asaccharide.
 10. The method of claim 9, wherein the saccharide isselected from the group consisting of glucose and trehelose.
 11. Themethod of claim 9, further comprising the step of c) freeze-drying thelyoprotected UCA, thereby preparing a freeze-dried UCA.
 12. The methodof claim 9, wherein the surfactant is selected from the group consistingof SPAN, alkylphenol ethoxylate-based surfactants, alcoholethoxylate-based surfactants, silicone-based surfactants, alkylpoly(ethylene oxide), alkylphenol poly(ethylene oxide), copolymers ofpoly(ethylene oxide) and poly(propylene oxide), alkyl polyglucosides,fatty alcohols, cocamide MBA, cocamide DEA, and polysorbates.
 13. Amethod of making a particle-stabilized ultrasound contrast agent (UCA),said method comprising the steps of: a) mixing at least two surfactantsin 50 ml of water where one of the surfactants is TPGS and heating saidmixture until both surfactants are dissolved; b) cooling said mixture toroom temperature while stirring rapidly until the dispersible waxy solidcomes out of solution as fine particles; e) purging the mixture using asterile filtered gas in an ice bath; d) sonicating the mixture atbetween 100-140 W for 1-5 minutes with constant purging; e) placingmixture in a separation funnel with 50 ml PBS to allow effectiveseparation of the bubbles; f) discarding lower 25 ml of the solution andtransferring the next 50-75 ml of solution remaining is placed in asecond separation funnel; and g) washing the bubbles of desired size,collecting them, and optionally, freeze drying them for storage.
 14. TheUCA of claim 3, wherein the surfactant is selected from the groupconsisting of SPAN, alkylphenol ethoxylate-based surfactants, alcoholethoxylate-based surfactants, silicone-based surfactants, alkylpoly(ethylene oxide), alkylphenol poly(ethylene oxide), copolymers ofpoly(ethylene oxide) and poly(propylene oxide), alkyl polyglucosides,fatty alcohols, cocamide MEA, cocamide DEA, and polysorbates.
 15. TheUCA of claim 3, wherein the UCA is a particle-stabilized UCA.
 16. TheUCA of claim 3, wherein the first surfactant is TPGS.